The present invention relates generally to the field of antibodies and altered antibodies, useful in the treatment and diagnosis of conditions mediated by IL-18, and more specifically to mAbs, Fabs, chimeric and humanized antibodies.
Human interleukin-18 is a recently identified cytokine that is synthesized as a biologically inactive 193 amino acid precursor protein (Ushio et al., J. Immunol. 156:4274, 1996). Cleavage of the precursor protein, for example by caspase-1 or caspase-4, liberates the 156 amino acid mature protein (Gu et al., Science 275:206, 1997; Ghayur et al., Nature 386:619, 1997), which exhibits biological activities that include the costimulation of T cell proliferation, the enhancement of NK cell cytotoxicity, the induction of IFN-xcex3 production by T cells and NK cells, and the potentiation of T helper type 1 (Th1) differentiation (Okamura et al., Nature 378:88, 1995; Ushio et al., J. Immunol. 156:4274, 1996; Micallef et al., Eur. J. Immunol. 26:1647, 1996; Kohno et al., J. Immunol. 158:1541, 1997; Zhang et al., Infect. Immunol. 65:3594, 1997; Robinson et al., Immunity 7:571, 1997). In addition, IL-18 is an efficacious inducer of human monocyte proinflammatory mediators, including IL-8, tumor necrosis factor-xcex1 (TNF-xcex1), and prostaglandin E2 (PGE2) (Ushio, S. et al., J. Immunol. 156:4274-4279, 1996; Puren, A. J. et al., J. Clin. Invest. 10:711-721, 1997; Podolin et al., J. Immunol. submitted, 1999).
The previously cloned IL-1 receptor-related protein (IL-1Rrp) (Parnet et al., J. Biol. Chem. 271:3967, 1996) was recently identified as a subunit of the IL-18 receptor (Kd=18 nM) (Torigoe et al., J. Biol. Chem. 272:25737, 1997). A second subunit of the IL-18 receptor exhibits homology to the IL-1 receptor accessory protein, and has been termed AcPL (for accessory protein-like). Expression of both IL-1 Rrp and AcPL are required for IL-18-induced NF-xcexaB and JNK activation (Born et al., J. Biol. Chem. 273:29445, 1998). In addition to NF-xcexaB and JNK, IL-18 signals through IL-1 receptor-associated kinase (IRAK), p56lck (LCK), and mitogen-activated protein kinase (MAPK) (Micallef et al., Eur. J. Immunol. 26:1647, 1996; Matsumoto et al., Biophys Biochem. Res. Comm. 234:454, 1997; Tsuji-Takayama et al., Biochem. Biophys. Res. Comm. 237:126, 1997).
Th1 cells, which produce proinflammatory cytokines such as IFN-xcex3, IL-2 and TNF-xcex2 (Mosmann et al., J. Immunol. 136:2348, 1986), have been implicated in mediating many of autoimmune diseases, including multiple sclerosis (MS), rheumatoid arthritis (RA), type 1, or insulin dependent, diabetes (IDDM), inflammatory bowel disease (IBD), and psoriasis (Mosmann and Sad, Immunol. Today 17:138, 1996). Thus, antagonism of a TH1-promoting cytokine such as IL-18 would be expected to inhibit disease development. Il-18 specific mAbs could be used as an antagonist.
The role of IL-18 in the development of autoimmune diseases has been demonstrated. Accordingly, it has been demonstrated that IL-18 expression is significantly increased in the pancreas and spleen of the nonobese diabetic (NOD) mouse immediately prior to the onset of disease (Rothe et al., J. Clin. Invest. 99:469, 1997). Similarly, IL-18 levels have been shown to be markedly elevated in the synovial fluid of rheumatoid arthritis patients (Kawashima et al., Arthritis and Rheumatism 39:598, 1996). Furthermore, it has been demonstrated that IL-18 administration increases the clinical severity of murine experimental allergic encephalomyelitis (EAE), a Th1-mediated autoimmune disease that is a model for multiple sclerosis. In addition, it has been shown that neutralizing anti-rat IL-18 antiserum prevents the development of EAE in female Lewis rats (Wildbaum et al., J. Immunol. 161:6368, 1998). Accordingly, IL-18 is a desirable target for the development of a novel therapeutic for autoimmunity.
Taniguchi et al., J. Immunol. Methods 206:107, describe seven murine and six rat anti-human IL-18 mAbs, which bind to four distinct antigenic sites. One of the murine mAbs (#125-2H), and the six rat mAbs inhibit IL-18-induced IFN-xcex3 production by KG-1 cells, with the rat mAbs exhibiting neutralizing activities 10-fold lower than that of #125-2H. As demonstrated by Western blot analysis, three of the murine mAbs, but none of the rat mAbs, are strongly reactive with membrane-bound human IL-18. In addition, an enzyme-linked immunosorbent assay (ELISA) to detect human IL-18 is described, utilizing #125-2H and a rat mAb. The limit of detection of this ELISA is 10 pg/ml.
European patent application EP 0 712 931 discloses two mouse anti-human IL-18 mAbs, H1 (IgG1) and H2 (IgM). As demonstrated by Western blot analysis, both mAbs react with membrane-bound human IL-18, but not with membrane-bound human IL-12. HI is utilized in an immunoaffinity chromatography protocol to purify human IL-18, and in an ELISA to measure human IL-18. H2 is utilized in a radioimmunoassay to measure human IL-18.
Neutralizing IL-18 antibodies may potentially be useful in relieving autoimmune diseases and related symptoms in man. Hence there is a need in the art for a high affinity IL-18 antagonist, such as a neutralizing monoclonal antibody to human interleukin 18, which would reduce Th1 differentiation and proliferation and thus autoimmune diseases and related symptoms.
In a first aspect, the present invention provides rodent (e.g., rat and murine) neutralizing monoclonal antibodies specific for human interleukin-18 and having a binding affinity characterized by a dissociation constant equal to or less than about 3.9xc3x9710xe2x88x9211 M as described in the detailed description. Exemplary of such monoclonal antibodies are the rat monoclonal antibody 2C10 and rat and murine monoclonal antibodies such as 14B7 and 13G9. Another aspect of the invention are hybridomas such as 19522C10(2)F2(1)A1, 195214B7(1)H10 and 187413G9(3)F12.
In a related aspect, the present invention provides neutralizing Fab fragments or F(abxe2x80x2)2 fragments thereof specific for human interleukin-18 produced by deleting the Fc region of the rodent neutralizing monoclonal antibodies of the present invention.
In still another related aspect, the present invention provides an altered antibody specific for human interleukin-18 which comprises complementarity determining regions (CDRs) derived from a non-human neutralizing monoclonal antibody (mAb) characterized by a dissociation constant equal to or less than about 3.9xc3x9710xe2x88x9211 M for human interleukin-18 and nucleic acid molecules encoding the same. When the altered antibody is a humanized antibody, the sequences that encode complementarity determining regions (CDRs) from a non-human immunoglobulin are inserted into a first immunoglobulin partner in which at least one, and preferably all complementarity determining regions (CDRs) of the first immunoglobulin partner are replaced by CDRs from the non-human monoclonal antibody. Preferably, the first immunoglobulin partner is operatively linked to a second immunoglobulin partner as well, which comprises all or a part of an immunoglobulin constant chain.
In a related aspect, the present invention provides CDRs derived from non-human neutralizing monoclonal antibodies (mAbs) characterized by a dissociation constant equal to or less than about 3.9xc3x9710xe2x88x9211 M for human interleukin-18, and nucleic acid molecules encoding such CDRs.
In still another aspect, there is provided a chimeric antibody containing human heavy and light chain constant regions and heavy and light chain variable regions derived from non-human neutralizing monoclonal antibodies characterized by a dissociation constant equal to or less than about 3.9xc3x9710xe2x88x9211 M for human interleukin-18.
In yet another aspect, the present invention provides a pharmaceutical composition which contains one (or more) of the above described altered antibodies and a pharmaceutically acceptable carrier.
In a further aspect, the present invention provides a method for treating conditions in humans associated with excess Th1 production, for example autoimmune diseases, by administering to said human an effective amount of the pharmaceutical composition of the invention.
In yet another aspect, the present invention provides methods for, and components useful in, the recombinant production of altered antibodies (e.g., engineered antibodies, CDRs, Fab or F(ab)2 fragments, or analogs thereof) which are derived from non-human neutralizing monoclonal antibodies (mAbs) characterized by a dissociation constant equal to or less than 3.9xc3x9710xe2x88x9211 M for human IL-18. These components include isolated nucleic acid sequences encoding same, recombinant plasmids containing the nucleic acid sequences under the control of selected regulatory sequences which are capable of directing the expression thereof in host cells (preferably mammalian) transfected with the recombinant plasmids. The production method involves culturing a transfected host cell line of the present invention under conditions such that an altered antibody, preferably a humanized antibody, is expressed in said cells and isolating the expressed product therefrom.
In yet another aspect of the invention is a method to diagnose conditions associated with excess Th1 production in a human which comprises obtaining a sample of biological fluid from a patient and allowing the antibodies and altered antibodies of the instant invention to come in contact with such sample under conditions such that an IL-18/antibody (monoclonal or altered) complex is formed and detecting the presence or absence of said IL-18/antibody complex.
Other aspects and advantages of the present invention are described further in the detailed description and the preferred embodiments thereof.